Immune-Related Circulating miR-125b-5p and miR-99a-5p Reveal a High Recurrence Risk Group of Pancreatic Cancer Patients after Tumor Resection

Abstract

Clinical follow-up aided by changes in the expression of circulating microRNAs (miRs) may improve prognostication of pancreatic ductal adenocarcinoma (PDAC) patients. Changes in 179 circulating miRs due to cancer progression in the transgenic Kras ^G12D/+; Trp53 ^R172H/+; P48-Cre (KPC) animal model of PDAC were analyzed for serum miRs that are altered in metastatic disease. In addition, expression levels of 250 miRs were profiled before and after pancreaticoduodenectomy in the serum of two patients with resectable PDAC with different progression free survival (PFS) and analyzed for changes indicative of PDAC recurrence after resection. Three miRs that were upregulated ≥3-fold in progressive PDAC in both mice and patients were selected for validation in 26 additional PDAC patients before and after resection. We found that high serum miR-125b-5p and miR-99a-5p levels after resection are significantly associated with shorter PFS (HR 1.34 and HR 1.73 respectively). In situ hybridization for miR detection in the paired resected human PDAC tissues showed that miR-125b-5p and miR-99a-5p are highly expressed in inflammatory cells in the tumor stroma, located in clusters of CD79A expressing cells of the B-lymphocyte lineage. In conclusion, we found that circulating miR-125b-5p and miR-99a-5p are potential immune-cell related prognostic biomarkers in PDAC patients after surgery.

Keywords: biomarkers; circulating microRNAs; immune cells; pancreatic cancer; progression free survival; resection.

Figure 1.

Overview of the serum miR screening in mice and patients to identify miRs associated with pancreatic cancer progression. (a) Schematic of performed RT-qPCR based miR discovery. Serum miRs from two groups of LSL-Kras^G12D/+; LSL-Trp53^R172H/+; P48-Cre (KPC) mice with different disease stages were analyzed for association with PDAC progression. Additionally, two patients with resectable PDAC were screened for expression of serum miRs to identify indicators of progression free survival (PFS) after surgical tumor removal. Overlapping differentially expressed miRs (DEmiRs) were selected for validation in an additional cohort of patients. (b) List of ≥3-fold DEmiRs that are associated with PDAC progression in KPC mice (left circle) and patients (right circle), identified in the miR screening. Upregulation of the miRs in bold red were associated with progressive PDAC in both mice and patients. These miRs were selected for validation.

Figure 2.

Pre- and post-resection expression levels of three serum miRs in PDAC patients with different PFS. Average expression levels of serum miRs 122–5p; 99a-5p or 125b-5p relative to the expression of two reference miRs, before and after surgery in three groups of patients. Short PFS in red = 0–8 months; median PFS in gray = 8–16 months; long PFS in green >16 months. Note the log scale of the y-axis. Error bars are SEM, P-values by one-way ANOVA comparing the three patient groups.

Figure 3.

MiR-125b-5p detection by in situ hybridization in pancreatic cancer tissues. Serial FFPE tissue sections were stained with H&E or a DIG labeled miR-125b-5p probe. MiR-125b-5p expression is detected by silver staining resulting in brown/black coloring, whereas nuclei are stained with hematoxylin in blue. (a) Representative images of low expression of miR-125b-5p, (green arrows), in untransformed pancreatic acinar cells and cells that underwent acinar to ductal metaplasia (ADM) or pancreatic intraepithelial neoplasia (PanIN). Scale bars are 100 μm in the normal acini and ADM; scale bar is 250 μm in the PanIN. (b) Representative images of cells in the tumor stroma with high expression of miR-125b-5p (red arrows). Corresponding tissue sections are stained with H&E or the B-lymphocyte lineage marker CD79A antibody by immunohistochemistry (brown). Scale bars are 100 μm in the top two rows; scale bars are 250 μm in the bottom row.

Figure 4.

MiR-99a-5p detection by in situ hybridization in pancreatic cancer tissues. Serial FFPE tissue sections were stained with H&E a DIG labeled miR-99a-5p probe, or the B-lymphocyte lineage marker CD79A antibody by immunohistochemistry (brown). Red arrows indicate miR-99a-5p expression, detected with silver staining resulting in brown/black coloring, whereas cell nuclei are stained with hematoxylin in blue. Scale bars are 250 μm in the first two columns; scale bar is 100 μm in the right-most column.